DMXL2 Antibody

What is DMXL2 and what are its key functions in cellular processes?

DMXL2, also known as Rabconnectin-3, functions as a scaffold protein for MADD and RAB3GA on synaptic vesicles, playing a crucial role in neuronal and endocrine homeostatic processes. It serves as a functional regulator of mammalian Notch signaling with WD domains and is abundantly expressed in the brain where it is enriched in synaptic vesicle fractions . Immunofluorescence and immunoelectron microscopy have revealed that DMXL2 is concentrated on synaptic vesicles at synapses . Recent research has demonstrated that DMXL2 can drive epithelial to mesenchymal transition in hormonal therapy resistant breast cancer through Notch hyper-activation .

What molecular weight should I expect when detecting DMXL2 in Western blots?

When performing Western blot analysis for DMXL2, you should expect to observe a band at approximately 340 kDa, which corresponds to the full-length protein (3036 amino acids) . Some researchers have also reported detecting an additional band at approximately 104 kDa in mouse tissue samples, which may represent a cleaved form or alternative isoform of the protein . Due to the high molecular weight of DMXL2, it is advisable to use low percentage (6-8%) or gradient gels with extended running times to achieve proper resolution.

What sample types and applications are most effective for DMXL2 antibody detection?

This data is compiled from multiple validated antibodies and represents optimal conditions based on published research .

What are the optimal conditions for immunohistochemical detection of DMXL2?

For optimal immunohistochemical detection of DMXL2 in paraffin-embedded tissues:

• Antigen retrieval: TE buffer pH 9.0 is strongly recommended, though citrate buffer pH 6.0 can serve as an alternative

• Primary antibody dilution: Most effective results are observed at dilutions ranging from 1:50 to 1:500, depending on the specific antibody

• Incubation conditions: Overnight incubation at 4°C typically yields optimal staining intensity and specificity

• Detection systems: Polymer-based detection systems provide superior sensitivity compared to conventional avidin-biotin methods

• Recommended tissues: Strong positivity has been documented in human testis, brain (particularly cerebellum), and kidney tissues

For paraffin-embedded human brain tissue, ab234771 has demonstrated effective staining at 1/100 dilution , while ab122552 has shown good results in human testis tissue at 1/50 dilution .

How should I optimize Western blot protocols for DMXL2 detection?

Optimizing Western blot protocols for DMXL2 requires specific considerations due to its high molecular weight:

• Sample preparation:

  • Include protease inhibitors to prevent degradation

  • Avoid repeated freeze-thaw cycles of samples

  • Heat samples at 70°C instead of 95°C to prevent aggregation of large proteins

• Gel electrophoresis:

  • Use 6-8% acrylamide gels or gradient gels (4-15%)

  • Extend running time at lower voltage (80-100V) to achieve better separation

  • Include high molecular weight markers (up to 400 kDa)

• Transfer conditions:

  • Employ wet transfer systems rather than semi-dry

  • Extend transfer time (overnight at 30V or 2-3 hours at 100V)

  • Consider adding SDS (0.1%) to transfer buffer to facilitate large protein transfer

• Antibody incubation:

  • For 27123-1-AP, use 1:500-1:1000 dilution

  • For 66891-2-Ig, use 1:2000-1:10000 dilution

  • Extend primary antibody incubation time (overnight at 4°C)

• Detection:

  • Use enhanced chemiluminescence systems with extended exposure times

  • Consider signal enhancers for high molecular weight proteins

These optimizations have proven effective across multiple cell lines (HEK-293, HeLa, Jurkat, MCF-7) and tissue samples (mouse and rat brain) .

What controls are essential when working with DMXL2 antibodies?

Implementing proper controls is critical for validating DMXL2 antibody specificity and experimental reliability:

• Positive controls:

  • Cell lines: Jurkat, HEK-293, HeLa, and MCF-7 cells show consistent DMXL2 expression

  • Tissues: Mouse brain, human testis, and human cerebellum demonstrate reliable DMXL2 detection

• Negative controls:

  • Primary antibody omission

  • Isotype-matched non-specific IgG

  • Pre-adsorption with immunizing peptide when available

• Validation controls:

  • DMXL2 knockdown/knockout samples (if available)

  • Comparison of staining patterns with multiple antibodies targeting different epitopes

  • Correlation of protein detection with known mRNA expression patterns

• Technical controls:

  • For WB: Loading controls appropriate for high molecular weight proteins (e.g., vinculin)

  • For IHC/ICC: Internal positive controls within the same tissue section

Implementing these controls ensures reliable and reproducible results when working with DMXL2 antibodies .

How can DMXL2 antibodies be utilized to investigate synaptic vesicle dynamics?

DMXL2's localization on synaptic vesicles makes it a valuable target for studying vesicle trafficking and dynamics:

• Co-localization studies:

  • Use dual immunofluorescence with DMXL2 antibodies (such as 66891-2-Ig at 1:400-1:1600) and established synaptic vesicle markers (e.g., synaptophysin, VAMP2)

  • Analyze co-localization coefficients using confocal microscopy and appropriate software

  • Examine spatial relationships between DMXL2 and active zone proteins

• Immunoprecipitation-based protein interaction analysis:

  • Use 24415-1-AP (0.5-4.0 μg per 1-3 mg lysate) to immunoprecipitate DMXL2 from neuronal lysates

  • Identify vesicle-associated binding partners through mass spectrometry

  • Validate interactions with known vesicle trafficking proteins (RAB3GA, MADD)

• Ultrastructural localization:

  • Employ immunogold electron microscopy with DMXL2 antibodies

  • Quantify the precise distribution of DMXL2 on synaptic vesicles at different stages of the vesicle cycle

  • Correlate DMXL2 localization with vesicle priming, docking, and fusion events

• Activity-dependent dynamics:

  • Compare DMXL2 distribution in resting versus stimulated neurons

  • Analyze potential phosphorylation-dependent relocalization following neuronal activity

These approaches leverage the specificity of validated DMXL2 antibodies to reveal mechanistic insights into synaptic vesicle function .

What methodologies can elucidate DMXL2's role in Notch signaling pathways?

To investigate DMXL2's function as a regulator of Notch signaling, researchers can employ several antibody-based approaches:

• Signaling pathway analysis:

  • Examine correlation between DMXL2 expression and Notch activation markers using dual immunofluorescence

  • Quantify nuclear translocation of Notch intracellular domain (NICD) in cells with varying DMXL2 levels

  • Analyze expression of Notch target genes in relation to DMXL2 expression patterns

• Protein-protein interaction studies:

  • Use co-immunoprecipitation with DMXL2 antibodies (e.g., 24415-1-AP) to identify interactions with Notch pathway components

  • Employ proximity ligation assays to visualize and quantify in situ interactions

  • Validate interactions through reciprocal co-immunoprecipitation experiments

• Functional manipulation:

  • Combine DMXL2 antibody detection with DMXL2 knockdown/overexpression systems

  • Correlate changes in DMXL2 levels with alterations in Notch pathway activation

  • Use DMXL2 antibodies to confirm target engagement in inhibitor studies

• Clinical correlation studies:

  • Analyze DMXL2 and Notch pathway component expression in tissue microarrays

  • Quantify correlation between DMXL2 levels and Notch activation in patient samples

  • Stratify outcomes based on co-expression patterns

These methodologies have proven particularly valuable in understanding DMXL2's role in driving epithelial to mesenchymal transition in hormonal therapy resistant breast cancer through Notch hyper-activation .

How do different DMXL2 antibodies perform in detecting potential post-translational modifications?

Detection of DMXL2 post-translational modifications (PTMs) requires careful antibody selection and experimental design:

When investigating DMXL2 PTMs:

• Phosphorylation analysis:

  • Use phospho-specific antibodies when available

  • Combine general DMXL2 detection with phosphatase treatments

  • Compare migration patterns before and after phosphatase treatment

• Glycosylation assessment:

  • Employ enzymatic deglycosylation (PNGase F, O-glycosidase) prior to Western blotting

  • Compare molecular weight shifts using antibodies targeting different epitopes

• Ubiquitination detection:

  • Perform DMXL2 immunoprecipitation followed by ubiquitin Western blotting

  • Use proteasome inhibitors to enhance detection of ubiquitinated forms

When using multiple antibodies targeting different regions, researchers can gain insights into domain-specific modifications that may regulate DMXL2 function .

How can I address inconsistent DMXL2 detection in Western blot applications?

Inconsistent detection of DMXL2 in Western blots can be addressed through systematic troubleshooting:

• Sample preparation optimization:

  • For brain tissue samples, use specialized extraction buffers containing mild detergents (0.5-1% NP-40 or Triton X-100)

  • Include higher concentrations of protease inhibitors than typically used

  • Consider using specialized high molecular weight protein extraction kits

• Electrophoresis and transfer adjustments:

  • For inconsistent high molecular weight detection (340 kDa band):

    • Reduce gel percentage to 6%

    • Extend running time by 30-50%

    • Use specialized transfer systems designed for high molecular weight proteins

  • For variable detection of lower bands (e.g., 104 kDa):

    • Optimize sample denaturation conditions

    • Test multiple antibodies targeting different epitopes

• Antibody selection and optimization:

  • 27123-1-AP has shown reliable detection in HEK-293, HeLa, Jurkat, MCF-7 cells and mouse brain tissue at 1:500-1:1000 dilution

  • 66891-2-Ig demonstrates consistent results in Jurkat cells and rodent brain tissues at 1:2000-1:10000 dilution

  • Compare results between antibodies to identify the most reliable option for your specific sample type

• Signal enhancement strategies:

  • Extend exposure times for chemiluminescent detection

  • Consider using signal enhancers specifically designed for high molecular weight proteins

  • Test alternative secondary antibodies with higher sensitivity

These systematic adjustments have resolved detection issues across multiple experimental systems .

Why might I observe different staining patterns with different DMXL2 antibodies in immunohistochemistry?

Different staining patterns observed with various DMXL2 antibodies in immunohistochemistry can be attributed to several factors:

• Epitope accessibility differences:

  • ab122552 targets amino acids 1900-2050, which may have different accessibility in fixed tissues compared to ab234771 targeting amino acids 2450-2700

  • Conformational changes during fixation can differentially affect epitope exposure

  • Different antigen retrieval methods may preferentially expose certain epitopes

• Antibody specificity profiles:

  • Some antibodies may detect specific DMXL2 isoforms or splice variants

  • Cross-reactivity with related proteins may occur with certain antibodies

  • Post-translational modifications might mask or expose specific epitopes

• Methodological variations:

  • Optimization table for addressing pattern discrepancies:

  Parameter	Adjustment Strategy	Expected Outcome
  Fixation	Test multiple fixatives (formalin, PFA, methanol)	May reveal fixative-sensitive epitopes
  Antigen retrieval	Compare TE buffer pH 9.0 vs. citrate pH 6.0	Different epitopes may require specific pH conditions
  Antibody concentration	Titrate each antibody independently	Determines optimal signal-to-noise ratio for each antibody
  Detection system	Compare polymer vs. avidin-biotin systems	May enhance detection of certain epitopes

• Validation approach:

  • Confirm specificity through peptide competition assays

  • Compare patterns with mRNA expression data (e.g., in situ hybridization)

  • Evaluate correlation with functional assays or known biological contexts

Understanding these factors helps researchers select the most appropriate antibody for their specific experimental question and tissue system .

What strategies can resolve weak signals in immunofluorescence detection of DMXL2?

To address weak signals when detecting DMXL2 by immunofluorescence:

• Sample preparation enhancement:

  • Optimize fixation conditions (4% PFA for 10-15 minutes shows good results for DMXL2)

  • Test different permeabilization methods (0.1-0.3% Triton X-100 for 5-10 minutes)

  • Implement antigen retrieval even for cultured cells (mild heat treatment in citrate buffer)

• Antibody optimization:

  • For ab234771, increase concentration from the standard 1/100 dilution

  • For 66891-2-Ig, use at the lower end of the recommended range (1:400) for weak signals

  • Extend primary antibody incubation time (overnight at 4°C rather than 1-2 hours)

• Signal amplification methods:

  • Implement tyramide signal amplification (TSA) for substantial signal enhancement

  • Use secondary antibodies with brighter fluorophores (Alexa Fluor 488 or 568 rather than FITC or TRITC)

  • Consider sequential application of multiple secondary antibodies

• Image acquisition adjustments:

  • Optimize exposure settings for specific signal range

  • Use confocal microscopy with spectral unmixing to reduce autofluorescence

  • Apply deconvolution algorithms to enhance signal-to-noise ratio

• Positive controls:

  • Include Jurkat cells as positive controls, which consistently show good DMXL2 staining with 66891-2-Ig

  • Use brain tissue sections as positive controls for tissue experiments

These comprehensive strategies have successfully resolved weak signal issues in multiple experimental systems .

How are DMXL2 antibodies being utilized to study neurological disorders?

DMXL2 antibodies are becoming increasingly important tools in neurological disorder research:

• Neurodegenerative disease studies:

  • Immunohistochemical analysis of DMXL2 expression in Alzheimer's and Parkinson's disease brain tissues

  • Comparison of DMXL2 synaptic vesicle localization between healthy and diseased samples

  • Correlation of DMXL2 levels with synaptic integrity markers in neurodegeneration

• Developmental neurological disorders:

  • Quantitative analysis of DMXL2 expression in neurodevelopmental disorder models

  • Investigation of DMXL2's role in neuronal migration and circuit formation

  • Assessment of potential alterations in subcellular localization

• Methodological approaches:

  • For human brain tissue analysis, ab234771 at 1/100 dilution has demonstrated effective staining

  • For rodent models, 66891-2-Ig in Western blots (1:2000-1:10000) and immunofluorescence (1:400-1:1600) provides sensitive detection

  • For protein interaction studies, 24415-1-AP is effective for immunoprecipitation from brain tissues

• Functional correlations:

  • Combined electrophysiological recordings with DMXL2 antibody staining

  • Assessment of DMXL2 levels in relation to synaptic dysfunction phenotypes

  • Correlation of DMXL2 expression with neuronal activity patterns

These approaches leverage DMXL2's role as a key controller of neuronal homeostatic processes and its importance in synaptic vesicle function .

What protocols are recommended for investigating DMXL2's role in cancer progression?

For investigating DMXL2's role in cancer progression, particularly in the context of therapy resistance and epithelial-to-mesenchymal transition:

• Expression analysis in clinical samples:

  • Immunohistochemical protocol:

    • Use ab122552 (1/50) or ab234771 (1/100) for paraffin-embedded human cancer tissues

    • Employ antigen retrieval with TE buffer pH 9.0

    • Quantify expression using digital pathology and scoring systems

  • Correlation with clinical parameters:

    • Hormone receptor status in breast cancer

    • Treatment response indicators

    • Patient outcome metrics

• Mechanistic studies in cell models:

  • Western blot analysis:

    • Use 27123-1-AP (1:500-1:1000) for detection in cancer cell lines

    • Compare expression between therapy-sensitive and resistant cell lines

    • Correlate with markers of Notch pathway activation

  • Immunofluorescence approach:

    • Use ab234771 (1/100) or 66891-2-Ig (1:400-1:1600) for cellular localization

    • Co-stain with EMT markers (E-cadherin, vimentin)

    • Analyze nuclear translocation of Notch signaling components

• Protein interaction studies:

  • Immunoprecipitation protocol:

    • Use 24415-1-AP (0.5-4.0 μg for 1-3 mg lysate)

    • Identify cancer-specific interaction partners

    • Validate interactions through reciprocal co-immunoprecipitation

• Functional validation:

  • Combine antibody detection with DMXL2 knockdown/overexpression

  • Monitor changes in Notch activation, EMT status, and therapy resistance

These protocols build on DMXL2's established role in driving epithelial to mesenchymal transition in hormonal therapy resistant breast cancer through Notch hyper-activation .

How can DMXL2 antibodies be integrated with cutting-edge research technologies?

Integration of DMXL2 antibodies with advanced research technologies is expanding our understanding of its functions:

• Single-cell analysis platforms:

  • Protocol for mass cytometry (CyTOF):

    • Metal-conjugate DMXL2 antibodies (typically 27123-1-AP or 24415-1-AP)

    • Combine with markers for cell type identification and signaling pathway activation

    • Analyze cellular heterogeneity in complex tissues

• Super-resolution microscopy applications:

  • STORM/PALM microscopy protocol:

    • Use directly labeled primary antibodies or high-quality secondary antibodies

    • ab234771 (1/100) has demonstrated good results in fluorescence applications

    • Resolve DMXL2 localization at nanoscale resolution

    • Assess co-localization with vesicle and signaling components

• Spatial transcriptomics/proteomics integration:

  • Multiplex immunofluorescence approach:

    • Combine DMXL2 antibody detection with spatial transcriptomics platforms

    • Use 66891-2-Ig (1:400-1:1600) for cellular visualization

    • Correlate protein expression with spatial gene expression patterns

• In vivo imaging applications:

  • Intravital microscopy protocol:

    • Employ fluorescently labeled Fab fragments of DMXL2 antibodies

    • Track dynamics in live animal models

    • Monitor responses to pharmacological interventions

These integrative approaches are at the forefront of DMXL2 research, combining antibody specificity with technological advances to reveal new biological insights .